Retinoid-dependent gene expression regulates early morphological events in the development of the murine retina.

Endogenous retinoids have been implicated in the axial patterning of the embryonic vertebrate retina; however, no studies have directly examined how asymmetric retinoid-dependent gene expression regulates early morphological events in the development of the retina. Here we used a line of indicator mice that possess a retinoid-dependent transgene to examine the relationship between retinoic acid (RA)-dependent gene expression and events occurring during early eye morphogenesis, such as the closure of the optic disc. We found that retinoid-regulated gene expression shifts along the dorsal/ventral axis of the embryonic retina; at embryonic day (E) E11.5 transgene expression is restricted to the neuroepithelium in dorsal retina, and by E14.5 only immature cells located in ventral retina and the dorsal retinal margins demonstrate transgene activation. By manipulating RA levels, we were not only able to systemically alter RA-dependent gene expression along the dorsal/ventral axis, but also to affect retinal morphology. In particular, reducing RA availability resulted in the abnormal closure of the optic fissure. These results indicate that asymmetric levels of RA regulate early RA-dependent gene expression in the eye and demonstrate that the normal pattern of retinoid-dependent gene transcription along the dorsal/ventral axis is critical for the proper development of the vertebrate retina.

Photoreceptor mosaic: number and distribution of rods and cones in the rhesus monkey retina.

Video-enhanced differential interference contrast optics was used to determine the number and distribution of photoreceptors across the entire retinal surface of 9 eyes obtained from 7 adult rhesus monkeys. We found that the retina of this primate contains an average of 3,100,000 cones (+/- 130,000) and 61,000,000 rods (+/- 7,500,000). Variation among animals in the number of rods and cones cannot be accounted for by differences in sex, age, or retinal surface area, nor is there a correlation between the number of rods and cones (a retina with a high number of rods does not typically have a high number of cones). Cone density peaks at 141,000 cones/mm2 in the foveola and decreases about 100-fold toward the periphery. Rod density in a central annulus around the fovea is 130,000/mm2 and decreases 6-8-fold toward the periphery. In all 9 retinae, we found that an area 4-5 mm dorsal to the fovea had the highest rod density at 184,000 rods/mm2. The functional significance of this area, which we term the dorsal rod peak (DRP), may be related to high sensitivity vision under scotopic conditions. Outside of the DRP, rod density is symmetrical around the major axes of the retina, whereas cone density is elevated in nasal retina. Among animals, both rods and cones display a 2-fold individual difference in receptor density at any given eccentricity. Although rods and cones differ in absolute number, the location and magnitude of their peak densities, and their central to peripheral density gradients, the ratio of the density of rods to cones (15-30:1) is remarkably stable from 3 mm to 15 mm eccentricity. The relative consistency in the proportion of rods and cones in extrafoveal retina may be related to mechanisms of retinal development and functional interactions between scotopic and photopic systems.

Positional information and opsin identity in retinal cones.

To test the hypothesis that local environmental cues regulate the expression of middle wavelength-sensitive (MWS) and short wavelength-sensitive (SWS) opsins in cone photoreceptors, we examined the development of the neonatal mouse retina in an organotypic culture system. The segregation of MWS and SWS cones into dorsal and ventral fields in the mouse retina offers an opportunity to isolate a phenotypically homogeneous population of immature cones prior to opsin expression. Retinae were harvested from mice ranging in age from birth (P0) to P18 and maintained in vitro for up to 4 weeks. Cones from newborn mice were first immunoreactive to SWS opsin-specific antibodies (OS-2 and JH455) after 5 days in vitro, which corresponds to a time course similar to that in vivo. The topographic separation of SWS cones into distinct dorsal and ventral fields was also obvious in retinal explants from newborn mice. However, the MWS opsin, identified by polyclonal antibody JH492, was expressed only in vitro when dorsal explants were harvested from P3 or older pups. Despite the absence of MWS opsin expression in newborn retinal cultures, there was no evidence of an increase in the numbers of SWS cones. To test if local diffusable cues could induce immature cones to express an aberrant opsin, dorsal and ventral retinal explants at different stages of maturation were cocultured during the incubation period. Neither the emergence of the cone fields nor the difference in the regional and temporal development of the MWS and SWS opsins was affected in these experiments. These results suggest that positional information in the retina and the opsin identity of cones is determined prior to birth and argue against the hypothesis that postnatal cones can be induced to express an aberrant opsin.

Duration of retinogenesis: its relationship to retinal organization in two cricetine rodents.

The Mongolian gerbil (Meriones unguiculatus) has a prolonged period of development relative to other muroid rodents. We have explored the consequences of this relatively long period of maturation on retinal cell number and topography by comparing the duration and topography of neurogenesis in the gerbil retina with that of a closely related species which develops rapidly, the Syrian hamster (Mesocricetus auratus) (Sengelaub et al.: J. Comp. Neurol. 246:527-543, 1986). An analysis of thymidine-labeled retinas indicate that cells destined for the gerbil retinal ganglion cell layer are generated for at least 12 embryonic days, twice the duration in the hamster. The period of cell loss in the gerbil retinal ganglion cell layer extends for at least 14 postnatal days, more than twice as long as in the hamster. The gerbil retina is generated in a center-to-periphery gradient for both retinal ganglion cells and displaced amacrine cells, while no such gradients are evident in the hamster retina. We conclude that the longer developmental period of the gerbil is associated with 1) a longer period of neurogenesis resulting in greater retinal cell number, 2) the expression of spatial gradients in neurogenesis, and 3) a larger eye at maturity. The last two factors, in part, may be related to the development of a highly differentiated area centralis and visual streak in the retina of this rodent. Unrelated to duration of growth, early differences in retinal shape between these two species contributes to the development of retinal topography. The gerbil, but not the hamster retina, is initially asymmetric, longer in its nasotemporal than its dorsoventral dimension. The gerbil retina then grows asymmetrically, producing a spherical retina, and coincident in time, a nasotemporally extended visual streak.

Early emergence of photoreceptor mosaicism in the primate retina revealed by a novel cone-specific monoclonal antibody.

We have examined the emergence of the photoreceptor mosaic in fetal macaque monkeys by using a novel monoclonal antibody, 7G6, that recognizes all cones in the adult primate retina. In the fetal retina, however, between embryonic (E) day 80 and E130, some opsin-positive cones were not labeled by 7G6. Double-labeling experiments revealed that though long and middle wavelength-sensitive fetal cones are 7G6-positive, a subset of short wavelength-sensitive cones are delayed in their acquisition of 7G6 immunoreactivity. Heterogeneity in cone labeling with 7G6 was evident at all fetal ages. The onset of 7G6 immunoreactivity, surprisingly, precedes both the expression of the cone opsins and the formation of synaptic contacts in the outer plexiform layer. Specifically, a population of cones was labeled in the periphery of the E65 retina, concomitant with ongoing cone genesis. Moreover, early-differentiating 7G6-positive cones are organized into a regular array in immature, peripheral regions of the fetal retina indicating that the spatial arrangement of cones is initiated either during active cone proliferation or the initial differentiation of these cells. These results suggest that the periodic spacing of cones in the primate retina emerges autonomously within the photoreceptor layer, prior to the formation of synaptic connections within the retina or with the brain.

Cone properties of retinal margin cells in the monkey (Macaca mulatta).

PURPOSE: To characterize a cell population in the monkey retinal margin that was labeled with a cone-specific antibody and to determine the presence of additional markers. METHODS: Retinal whole-mount preparations from infant and adult rhesus monkeys (Macaca mulatta) were immunolabeled by incubation overnight with the primary antibodies 7G6, a cone-specific antibody; SV2, a synaptic-vesicle antibody; and opsin antibodies that recognize either the short or long/middle wavelength-sensitive opsins. RESULTS: The retinal margin cells labeled by 7G6 lay within 1 mm of the ora serrata and differed from 7G6-labeled cones in the central retina. The margin cells possessed a soma, a fiber process, and a terminal enlargement that lay in the plane of the retina; no outer segment was discernible. A total of 5400 and 7252 margin cones cells were found in each of two monkeys. The terminal enlargement and soma of the labeled margin cells also showed SV2 immunoreactivity. Surprisingly, opsin immunoreactivity extended throughout the margin cell, which is consistent with the absence of a discernible outer segment. CONCLUSIONS: Cells with immunoreactive cone properties were found in the margin of the monkey retina. The absence of an outer segment and the presence of somatic opsin and SV2 are reminiscent of features observed in the central cones of fetal monkey retinas. These results suggest that a subpopulation of cones in the retinal margin might fail to mature completely and thus retain juvenile characteristics into adulthood.

The subcellular localization of Otx2 is cell-type specific and developmentally regulated in the mouse retina.

Recent evidence implicates homeodomain-containing proteins in the specification of cell fates in the central nervous system. Here we report that in the embryonic mouse eye Otx2, a paired homeodomain transcription factor, was found in retinal pigment epithelial cells and a restricted subset of retinal neurons, including ganglion cells. In the postnatal and adult eye, however, both the cellular and subcellular distribution of the Otx2 protein were cell type-specific. Otx2 was detected only in the nuclei of retinal pigment epithelial and bipolar cells, but was present in the cytoplasm of rod photoreceptors. Immunohistochemical studies of retinal explants and transfected cell lines both suggested that the retention of Otx2 in the cytoplasm of immature rods is a developmentally regulated process. The differential distribution of Otx2 in the cytoplasm of rods and the nucleus of other cell types, suggests that subcellular localization of this transcription factor may participate cell fate determination during specific phases of retinal development.

Aromatase in developing sensory systems of the rat brain.

Sex differences in the rat brain are dependent, in part, on oestrogen exposure during specific developmental perinatal periods. The availability of oestrogen requires precursor androgen and the presence of intraneuronal aromatase. To examine sites of oestrogen formation and action in the brain, immunocytochemical and biochemical localization of aromatase in the rat brain were determined between embryonic day 14 and postnatal day 20. Aromatase-immunolabelled neuronal profiles were present in hypothalamic, cortical and limbic regions. Surprisingly, aromatase immunoreactivity was also observed in non-limbic regions of the immature brain where it was previously unsuspected. Among these regions, aromatase staining was robust in developing sensory systems, including primary afferents of the olfactory, trigeminal, vestibulocochlear, and visual systems. To determine whether this aromatase is functional in these systems, i.e. converts testosterone to estradiol, the trigeminal nerve was dissected from the hindbrain of perinatal animals and studied for enzyme activity by the tritium release method. The dpm/mg protein/h tritium release in these tissues equalled that of hypothalamic or limbic controls, indicating that these sensory areas are sites of in-situ estradiol synthesis. Our data suggests that aromatase (estradiol)-dependent mechanisms may play a role in the differentiation and maturation of sensory pathways, which, in turn, may contribute to sex differences in the activity of these systems.

Temporal retina is preferentially represented in the early retinotectal projection in the hamster.

Retrograde transport of horseradish peroxidase (HRP) after complete transection of the brachium of the superior colliculus on the day of birth in hamsters revealed preferential labelling of the temporal retina. Cytochrome oxidase staining of the retina showed similar preferential temporal labelling. A discrete lack of label of the extreme temporal periphery of the retina contralateral to the HRP placement and a complementary label of ipsilateral temporal periphery were also observed.

Control of cell number in the developing visual system. II. Effects of partial tectal ablation.

The effects of potential excess innervation on cell survival in the superior colliculus and related structures during the period of normally occurring cell death was examined. A unilateral, partial lesion of the superficial layers of the superior colliculus on the day of birth, which results in a compression of the retinotectal map into the remaining area, was the manipulation used to produce the potential excess innervation. Cell density was reduced in the tectal fragment early in development, consistent with hyperinnervation, but had returned to normal by the end of the period of normally occurring cell death. The overall incidence of cell degeneration in the remaining partial colliculus was not different from the undamaged contralateral colliculus or from normal, though there was evidence of a transitory depression and later elevation of cell loss. Cell loss in the retina contralateral to the lesion was increased in the late part of the period of normal cell loss and there were fewer cells in the retinal ganglion cell layer at maturity. The amount of the cell loss in the retina was small compared to the amount of target removal. These results suggest that the survival of neurons with branching axons does not sensitively reflect target availability.

Relation of an array of early-differentiating cones to the photoreceptor mosaic in the primate retina.

The retina of diurnal primates, including humans, contains a reiterative mosaic of red-, green- and blue-sensitive cones whose visual pigments are maximally sensitive to long, middle or short wavelengths, respectively. Although the distribution of the cone subtypes in the adult rhesus monkey has been quantified using opsin-specific antisera, the mechanism for the phenotypic specification of the cone subtypes and the establishment of their ratios in the retinal mosaic remain unknown. Here we present immunocytochemical evidence that a subset of cones (about 10%) express their cell-specific opsin two to three weeks before the surrounding cones. Remarkably, these precocious cones are evenly stationed throughout undifferentiated regions of the retinal surface from several weeks after their last mitotic division, and at least one month before the formation of their synapses with bipolar and horizontal cells. Use of confocal laser microscopy reveals that the inner segments of immunolabelled and surrounding unlabelled cones are transiently in apposition with one another, enabling surface mediated interactions to occur during this period. We suggest that the early maturing cones induce neighbouring undifferentiated cones to express an appropriate opsin phenotype, and therefore constitute a 'protomap' for the emergence of the species-specific retinal mosaic.

Development of photoreceptor mosaics in the primate retina.

The mosaic of rods and cones in the primate retina is the neuronal array where the visual world is first mapped onto the central nervous system. Rods, which mediate scotopic vision, and cones, which mediate photopic and color vision, are found in all vertebrate species. However, regional differences in the topographic arrangement and ratio of rods to cones vary dramatically among species, including different primates. Furthermore, the proportion and distribution of the wavelength-sensitive cone subtypes vary considerably between primates that occupy different visual habitats. What genetic or environmentally regulated mechanisms specify the position, phenotype, and ratios of photoreceptor subtypes? Available data suggest that the transient appearance of early-differentiating cones may serve to delineate basic species-specific retinal coordinates and determine the opsin phenotype of local assemblies of cones in the fetal photoreceptor mosaic. This article will summarize presently available data and our ideas of how the photoreceptor mosaic is organized in the adult primate retina, the features of these mosaics, which vary between primate species, and the developmental mechanisms, which may account for the emergence of photoreceptor position and specification of their phenotypes in the primate retina.

An array of early differentiating cones precedes the emergence of the photoreceptor mosaic in the fetal monkey retina.

We previously have demonstrated that approximately 10% of cones in the fetal monkey retina precociously express the red/green opsin. These data suggested the possibility that a subset of cones differentiates prior to their nascent cone neighbors. To further assess this early cone differentiation in the fetal monkey retina, we used monoclonal antibodies proven to be important developmental markers of photoreceptor phenotypes and synaptogenesis (XAP-1, specific to photoreceptor membranes; SV2, specific to synaptic vesicle protein). Although these two antibodies recognize functionally distinct antigens, our analyses revealed that both identify a subset of precociously immunoreactive cones. Further, XAP-1- and SV2-positive cones are distributed in the same pattern as precocious red/green-sensitive cones in immature regions of the fetal monkey retina. These results support the hypothesis that the primate retina possesses a spatially organized protomap that may induce the emergence of the photoreceptor mosaic and trigger the formation of color-specific pathways that include horizontal, bipolar, and retinal ganglion cells.

Distribution of photoreceptor subtypes in the retina of diurnal and nocturnal primates.

We have used antibodies specific to either the red/green-or blue-sensitive cones in order to compare their ratio and distributions to that of the rods in the retinae of 3 primate species that differ in their capacity for color vision. We have found that the monoclonal antibody CSA-1 (Johnson and Hageman, 1988) and the polyclonal antibody 4942A, specific to the red- and green-cone opsin (Lerea et al., 1989), applied to retinal whole-mounts labeled approximately 90% of all cones in the diurnal Old-World rhesus monkey (Macaca mulatta) and all of the cones in the nocturnal New-World owl monkey (Aotus trivirgatus) and nocturnal prosimian bushbaby (Galago garnetti). The polyclonal antibody 108B, specific to the blue-cone opsin (Lerea et al., 1989), labeled about 10% of the cones across the entire surface of the rhesus monkey retina, but failed to label any cones in the retina of the 2 nocturnal species. Only the retina of the rhesus monkey possessed an all-cone foveola in which the density of cone inner segments was 17-fold greater than that in the fovea of the owl monkey or bushbaby retina. Surprisingly, the density of cones per unit area outside of the fovea was comparable in all 3 species. Rod density in the dorsal retina was elevated in all animals examined, but was 2-3 times greater in the nocturnal species than in the rhesus monkey retina. Application of the photoreceptor-class-specific antibodies may provide further insights into the evolution and development of wavelength sensitivity in the retina, as well as enhance our understanding of normal and abnormal color vision in humans.

Development of ganglion cell topography in ferret retina.

The adult ferret has approximately 90,000 retinal ganglion cells, arranged in a prominent area centralis and visual streak. The role of differential cell generation, cell death, and retinal growth in the control of adult retinal ganglion cell number and distribution was evaluated by examining basic aspects of retinogenesis, including growth in retinal area, developmental changes in the number, size, and distribution of retinal ganglion cells (identification aided by retrograde transport of HRP), and the incidence of degenerating cells in the ganglion cell layer. Retinal development in the ferret was also compared to retinal development in the cat (which has an even more differentiated area centralis) to determine what alterations of developmental parameters are most closely associated with this species difference in adult morphology. The area of the retina increases linearly from birth (12 mm2) to postnatal day 24 (54 mm2), reaching an eventual adult value of 64 mm2. Ganglion cell numbers peak at 155,000 (approximately twice the adult number) on postnatal day 3, and fall to adult numbers by postnatal day 6. The remaining cells of the ganglion cell layer, principally displaced amacrine cells, reach their peak number on postnatal day 10 (approximately 280,000), falling to 200,000 by adulthood. Degenerating cells are abundant in the ganglion cell layer in the immediate postnatal period. A difference in the incidence of degenerating cells in the presumptive area centralis versus that in the retinal periphery was not observed postnatally, though there were other striking spatial nonuniformities, suggesting that differential cell loss might contribute to other features of retinal topographic organization. Ganglion cell density is virtually uniform across the retina at birth. Cell density is first reduced in the dorsal retina, resulting in a dorsal-to-ventral gradient in cell density that persists until day 10, when ganglion cell number has stabilized. By postnatal day 24, an area centralis and visual streak has emerged, but not of adult magnitude. Because ganglion cell number has stabilized long before the area centralis and visual streak emerge, we conclude that differential retinal growth is the principal mechanism producing this feature of retinal topography. Comparison with the cat suggests that the proportionately greater nonuniform growth of the cat's eye accounts for the greater differentiation of its area centralis.

Regressive events in brain development and scenarios for vertebrate brain evolution.

The problems of the evolution of varying brain size, the specialization of particular functional systems and overall differences in the relative complexity of brain organization are discussed in terms of alterations of regressive events in neurogenesis (cell death and axon retraction). Three scenarios for evolution, cascade reorganization, parcellation and heterochrony, are considered in light of regressive mechanisms during development.

Early divergence of magnocellular and parvocellular functional subsystems in the embryonic primate visual system.

In both human and Old World primates visual information is conveyed by two parallel pathways: the magnocellular (M) and parvocellular (P) streams that project to separate layers of the lateral geniculate nucleus and are involved primarily in motion and color/form discrimination. The present study provides evidence that retinal ganglion cells in the macaque monkey embryo diverge into M and P subtypes soon after their last mitotic division and that optic axons project directly and selectively to either the M or P moieties of the developing lateral geniculate nucleus. Thus, initial M projections from the eyes overlap only in prospective layers 1 and 2, whereas initial P projections overlap within prospective layers 3-6. We suggest that the divergence of the M and P pathways requires developmental mechanisms different from those underlying competition-driven segregation of initially intermixed eye-specific domains in the primate visual system.

Differential elasticity of the immature retina: a contribution to the development of the area centralis?

Differential stretch of a retinal surface with an initially uniform cell density has been repeatedly implicated as one of the developmental mechanisms that produces the topographic organization of cell density in the adult retina, notably the area centralis or visual streak versus peripheral regions. It is known that intraocular pressure is required to produce the normal conformation and thinning of the retina during development. We tested the possibility that the retina has elastic properties that might permit differential stretch in conjunction with intraocular pressure. The relative deformation of the retina containing the presumptive area centralis was compared to the deformation of peripheral retina at equivalent applied fluid displacements in 7-12-day-old cats. The peripheral retina deformed significantly more, consistent with the hypothesis that differences in the local elasticities of the developing neural retina contribute to its characteristic topographic changes. Thus, a biomechanical property of the growing eye may contribute to the mechanism by which the pattern of the visual array is sampled.

Localization of protein kinase C to UV-sensitive photoreceptors in the mouse retina.

The present study has identified a population of cone photoreceptors in the murine retina that are uniquely immunoreactive for protein kinase C (PKC). Wavelength-sensitive cone subtypes are segregated along the dorso-ventral axis in the mouse retina with ventral retina occupied exclusively by ultraviolet wavelength-sensitive (UVWS) cones, and dorsal retina dominated by middle wavelength-sensitive cones. PKC-positive cones are found primarily in the ventral retina, and double-label immunocytochemistry using a short wavelength-sensitive opsin antibody confirms that they specifically correspond to the UVWS cone subtype. The PKC antibody, as documented in other mammals, also identifies rod bipolar cells in the mouse retina. UVWS cones and bipolar cells have previously been shown to share transcriptional regulatory elements, as observed in transgenic mice encoding a portion of the human SWS-opsin promoter controlling the lacZ reporter gene. In such mice, the transgene product, beta-galactosidase, is expressed in populations of both cones and bipolar cells. The present study confirms that lacZ-expressing photoreceptors are indeed PKC-positive photoreceptors, but that the lacZ-expressing bipolar cells are not the PKC-positive rod bipolar cells. These cells must correspond to a type of cone bipolar cell.